1. Field of Invention
The present invention relates to analytical methods for the detection of cis-diol containing compounds including catecholamines. More specifically, the present invention provides a quantitative assay for detecting dopamine, epinephrine or norepinephrine in plasma of individuals using mass spectrometry.
2. Description
Catecholamines (dopamine, norepinephrine/noradrenaline, and epinephrine/adrenaline) act as neurotransmitters or hormones at both central and peripheral levels, in both neuronal and non-neuronal tissues. The involvement of catecholamines in multiple regulatory systems and metabolic processes supports their important role as biomarkers for the clinical diagnosis, therapy, and prognosis of several neuroendocrine and cardiovascular disorders.
Tyrosine is the amino acid precursor of catecholamines which produces L-dihydroxylphenyl alanine (L-DOPA) through the action of tyrosine hydroxylase. In the central nervous system the activity of decarboxylase activity with L-DOPA produces dopamine and norepinephrine which act as neurotransmitters. The adrenal medulla produces both epinephrine and norepinephrine (via dopamine β-hydroxylase). The neurons release norepinephrine through postganglionic sympathetic nerves. Conversion of norepinephrine to epinephrine occurs mainly in the adrenal medulla through the activity of N-methyl transferase. FIG. 1 provides the molecular structure and physiochemical properties for dopamine, noradrenaline, and adrenaline.
When catecholamines are released from the sympathetic tissue, other than the adrenal medulla, the primary means of physiologic inactivation is the return of unaltered catecholamines by an active transport mechanism. The residual hormone may then be metabolized or excreted unchanged by the kidney.
When metabolized, two enzymes are important, monamine oxidase (MAO) which is responsible for oxidative deamination and catechol-O-methyltransferase (COMT), which is responsible for O-methylation. COMT is principally responsible for inactivating circulating catecholamines, whereas MAO is thought to play a role in disposing of excess catecholamine stores. The major end product for metabolism of epinephrine or norepinephrine is 3-methoxy-4-hydroxymandelic acid (VMA). Other urinary metabolites of catecholamines are metanephrine and normetanephrine.
Catecholamines have very low molecular weights and are extremely low concentration in biological samples. Analysis of catecholamines in biological sample is prone to a lot of potential interference present in biological matrix. Catecholamines are extremely polar and very weakly retained chromatographically in commonly used reverse phase liquid chromatography. Catecholamines are chemically unstable, prone to spontaneous oxidation and decompose easily at high pH. Catecholamines chelate quickly to multivalent cation such as iron, copper, etc. in solution. Catecholamines adsorb strongly to various contact surfaces such as glass and metal by complex formation, including fluidic path in the LC-MS/MS system, leading to significant absorption loss. The measurement of catecholamines in biological samples remains a current analytical challenge, in spite of the great diversity of methodologies that have been developed throughout the years. The quantification of catecholamines in biological samples demands specific and very sensitive bioanalytical methods.
Current standard method of choice is high performance liquid chromatography (HPLC) with electrochemical (ECD) detection, despite time consuming sample preparation, long chromatographic runtime (approximately 30 minutes), prone to various interferences, and low sensitivity, severely limiting its clinical use. Other methods include fluorometric assays, radio enzymatic assays (REA), gas chromatography with mass spectrometric detection (GC-MS), radio immunoassays (RIA) and enzyme immunoassays (EIA). Fluorometric and radio enzymatic assays lack accuracy and reproducibility and the fluorometiric assays do not differentiate between different catecholamines. HPLC and GC-MS are time consuming, need complex technical equipment and, therefore, are cost-intensive, leaving any suitable methods for determining catecholamines based on reliability, cost, accuracy, reproducibility and rapidness to be immunoassays.
In currently available EIA or RIA based assays for determining catecholamines, the catecholamines are first extracted out of the sample, then derivatized for antibody recognition and to enhance their stability, and thereafter detected using antibodies specific for catecholamines. Consequently, the most commonly used extraction method is binding of the catecholamines via their cis-diol structure to a boronate affinity gel (Cannizzo C. et al., 2005 Polymer 46, 1269-1276). The subsequent modification to stabilize the catecholamines is done using the enzyme catechol-O-methyl-transferase (COMT) which O-methylates the 3-hydroxyl group of the catechol moiety. For the COMT to access the 4-hydroxyl group of the catechol moiety, the catecholamines have to be released from the affinity gel which is achieved by adjusting the pH to acidic conditions. However, COMT is not active under acidic conditions. Therefore, in order to allow the reaction catalyzed by COMT to take place and prevent rebinding of the catecholamines released from the boronate affinity gel, the released catecholamines have to be separated from the boronate affinity gel by transferring them into another reaction vessel. This transfer of the sample creates an additional source of error and complicates the assay.
One means for reducing the transfer steps in methods for analyzing catecholamines by EIA is to perform the enzymatic O-methylation and the subsequent EIA in one reaction vessel. However because derivatization of the catecholamines and binding to the specific antibodies proceed in a parallel manner and since only derivatized catecholamines are detected by the antibodies, the results and kinetics of the EIA are less precise. Further, this method cannot be performed in standard automated EIA analyzer due to the preceding enzymatic reaction taking place in the same reaction vessel.
The assay of the present invention provides a simple, fast, and sensitive 2D-LC-MS/MS method to measure three catecholamines (dopamine, norepinephrine, and epinephrine) in a routine clinical laboratory setting.